www.nature.com/scientificreports OPEN A model‑based quantifcation of startle refex habituation in larval zebrafsh Carolina Beppi1,2,3,4*, Dominik Straumann1,2,3,4,5 & Stefan Yu Bögli2,3,4,5 Zebrafsh is an established animal model for the reproduction and study of neurobiological pathogenesis of human neurological conditions. The ‘startle refex’ in zebrafsh larvae is an evolutionarily preserved defence response, manifesting as a quick body‑bend in reaction to sudden sensory stimuli. Changes in startle refex habituation characterise several neuropsychiatric disorders and hence represent an informative index of neurophysiological health. This study aimed at establishing a simple and reliable experimental protocol for the quantifcation of startle refex response and habituation. The fsh were stimulated with 20 repeated pulses of specifc vibratory frequency, acoustic intensity/power, light‑intensity and interstimulus‑interval, in three separate studies. The cumulative distance travelled, namely the sum of the distance travelled (mm) during all 20 stimuli, was computed as a group‑level description for all the experimental conditions in each study. Additionally, by the use of bootstrapping, the data was ftted to a model of habituation with a frst‑order exponential representing the decay of locomotor distance travelled over repeated stimulation. Our results suggest that startle habituation is a stereotypic frst‑order process with a decay constant ranging from 1 to 2 stimuli. Habituation memory lasts no more than 5 min, as manifested by the locomotor activity recovering to baseline levels. We further observed signifcant efects of vibratory frequency, acoustic intensity/power and interstimulus‑interval on the amplitude, ofset, decay constant and cumulative distance travelled. Instead, the intensity of the fashed light did not contribute to signifcant behavioural variations. The fndings provide novel insights as to the infuence of diferent stimuli parameters on the startle refex habituation and constitute a helpful reference framework for further investigation. Zebrafsh has become an established animal model due to several advantages, including its small size, the prosper- ous ofspring, the rapid development through defned stages, the simple breeding and the easy maintenance of large stocks1,2. Te existence of established microscopic imaging approaches3–8 and the optical translucency until larval stages1,6,7 allow detailed non-invasive tracking of CNS changes following specifc neural manipulations or chemical interventions. Cell-manipulations (e.g., in Bhandiwad et al.9) and electrophysiological recordings can assess the zebrafsh sensory-processing functions fast and reliably 10–13, although invasively. Behavioural screen- ings are therefore becoming preferred assessment-methods of sensory-processing and attention14. Habituation of the startle refex: a basic form of learning. Zebrafsh at larval stage already exhibit a vast behavioural repertoire that can be reliably tracked and consistently quantifed15–19. First probed in the 1970s20, the startle refex in zebrafsh larvae is a fast body muscle contraction in response to abrupt sensory stimuli3,21,22 and is an evolutionarily preserved mechanism of defence against threat. It is a ‘C-bend’ of the body 23 largely mediated by the Mauthner cells (MC), a bilateral pair of large reticulospinal interneurons in the zebrafsh hindbrain22–26. Tey receive input ipsilaterally, through their lateral dendrites, from sensory receptors of difer- ent modalities24–27, although the mechanosensory hair cells are the main driver24,26. Transient light stimuli evoke increased escape responses kinematically identical to acoustic startle refex (ASR) C-bends and proportional to the magnitude of change in lighting21, but of a longer latency (M = ~ 183 ms) compared to acoustic and touch-evoked (M ~ 15 ms) startle responses21. Te startle refex can also be non-MC 1Neuroscience Center Zurich, University of Zurich and ETH Zurich, CH-8091 Zurich, Switzerland. 2Department of Neurology, University Hospital Zurich and University of Zurich, CH-8091 Zurich, Switzerland. 3Clinical Neuroscience Center, University Hospital Zurich and University of Zurich, CH-8091 Zurich, Switzerland. 4Swiss Concussion Center, Schulthess Clinic, CH-8008 Zurich, Switzerland. 5These authors contributed equally: Dominik Straumann and Stefan Yu Bögli. *email: [email protected] Scientifc Reports | (2021) 11:846 | https://doi.org/10.1038/s41598-020-79923-6 1 Vol.:(0123456789) www.nature.com/scientificreports/ initiated, although it has a slower onset28. Startle responses are modulable by internal factors like social domi- nance (in adult fsh), arousal or stress29–31. Increase in the startle refex can be induced by sensitisation, fear- potentiation32, and pharmacological compounds afecting the methyl-D-aspartic acid signalling33,34, while dec- remented ASR can be caused by pre-pulse inhibition35,36 and by habituation35. Habituation is a basic, non-associative, form of learning defned by the cessation of an innate response afer repeated or sustained exposure to a sensory stimulus 28,37,38. It has been studied in several invertebrate species, including Aplysia, Tritonia, Drosophila, as well as rodents, with a variety of stimuli 39–42. Te habituation of the startle refex in larval zebrafsh has been probed in the 1970s24,43. It is consistently elicitable and quantif- able, although non-stereotypic29–31. Te amount of stimulation the fsh is exposed to determines the endur- ance of habituation 28. Specifcally, three types of habituation persistence have been described: rapid habituation (≤ 15 min) is induced by low-frequency stimuli, while short-term habituation (≤ 1 h) can be induced by a few blocks of stimuli. If the blocks are repeated over several sessions, habituation can also endure for days (long- term habituation). Changes in startle refex and habituation have been reported in neuropsychiatric disorders, including schizophrenia44, traumatic brain injury and post-traumatic stress disorder 45,46. Te startle refex and habitu- ation are thus becoming established neurophysiological indices for clinical and translational investigation in neurology and psychiatry. Aims and hypotheses. A number of studies report that the number, duration and intensity of the stimuli, as well as the inter-stimuli interval and the number of blocks or sessions can afect the profle of the startle refex habituation19,28,35,47–49. However, the considerable variability in the experimental design and analytical approach of existing studies do not allow a direct comparison between the fndings, preventing the possibility to draw conclusions about the optimal experimental parameters. Te acquisition of complex data is one of the factors that may hinder the replicability and reproducibility of the fndings, which is a recurrent issue in behavioural (neuroscience) research in zebrafsh 50. Tis investigation aimed at defning a novel experimental protocol for the study and assessment of startle refex and its habituation. Specifcally, we aimed at designing a simple behav- ioural paradigm that could be more easily reproducible and adaptable for testing diferent experimental ques- tions, thus allowing to understand the infuence of specifc experimental parameters on the profle of the startle refex and its habituation. We investigated startle refex habituation in three separate studies, each manipulating and assessing the isolated efect of diferent stimulus features on the profle of the startle refex (acoustic or visual) habituation. A frst study (1) investigated the time necessary for the zebrafsh to extinguish the habituation, quantifying the ASR habituation at diferent re-test times relative to the baseline test. Based on Roberts et al.28, we hypoth- esised that startle extinction of ASR habituation memory, quantifed as the mean of all fsh’s cumulative distance travelled (CDT) during all 20 stimuli, would be signifcantly diferent from baseline, at the diferent re-test times (1, 5 or 15 min). Te second study (2) investigated the main efect of vibratory power (a), vibratory frequency (b), interstimulus interval (ISI) duration (c) on the profle of the ASR habituation. Te third study (3) assessed the efect of light-fash illuminance on the profle of the visual startle refex (VSR) habituation. Based on previ- ous fndings19,28,35,47–49, we further hypothesised that startle refex habituation would difer signifcantly between vibratory frequency conditions, acoustic power, ISI duration and illuminance. Results Study 1—Extinction of ASR habituation memory. Tis study was conducted to determine the decay of habituation memory, namely the time necessary for the fsh to ‘lose memory’ of the stimulus, as manifested by the locomotor responses to the stimulus returning up to baseline levels. It was hypothesised that in each re-test time group, there would be a statistically signifcant diference in average CDT at re-test compared to baseline. Wilcoxon signed-rank t-tests (N = 48) indicated that the median CDT rank at baseline was signifcantly higher compared to at 1 min re-test (Z = 202, p < 0.001), but not at 5 min re-test (Z = 469, p = 0.58) or at 15 min re-test (Z = 468, p = 0.31), as shown in Fig. 1G–I. Te mean distance travelled at each stimulus, by each group, is depicted in Fig. 1A–F. Descriptive statistics for the mean CDT values of all groups are reported in Table 1 (study 1). Te response probability was: 85% and 75% for the 1 min baseline and re-test conditions respectively, 77% and 67% for the